Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct
Background: Interferon beta (IFNβ) is one of the important cytokines expressed in response to stimulating factors such as antigens and plays roles in immunity and inflammatory process. In present study, the expression level of IFNβ-1a was examined in HEK293 cell line using real-time polymerase chain...
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| Format: | Article |
| Language: | fas |
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Isfahan University of Medical Sciences
2015-12-01
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| Series: | مجله دانشکده پزشکی اصفهان |
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| Online Access: | http://jims.mui.ac.ir/index.php/jims/article/view/5030 |
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| author | Raheleh Norouzi Zohreh Hojati |
| author_facet | Raheleh Norouzi Zohreh Hojati |
| author_sort | Raheleh Norouzi |
| collection | DOAJ |
| description | Background: Interferon beta (IFNβ) is one of the important cytokines expressed in response to stimulating factors such as antigens and plays roles in immunity and inflammatory process. In present study, the expression level of IFNβ-1a was examined in HEK293 cell line using real-time polymerase chain reaction (Real-Time PCR).
Methods: IFNβ gene sequence was amplified using specific primers contained KpnI and BglII restriction site from pSVMdhfr-IFNβ plasmid as template. It was cloned in similarly digested pBud.CE4.1 linear vector. Construction of recombinant plasmid was verified via restriction fragment length polymorphism (RFLP) analysis, colony PCR and gene sequencing. Recombinant plasmid was transformed into competent Escherichia coli Top10 cells finally. After amplification, recombinant plasmid was purified and transfected into HEK293. At last, RNA extraction, cDNA synthesis and analysis of expression level of gene were performed using Real-Time PCR method.
Findings: IFNβ gene was expressed under eEf1a promoter in HEK293 successfully. The expression level of target gene was increased 79.9 times in comparison with the control via transfection. Transfection of null vector showed 2.87 times elevation of target gene expression in response to the alien genome entered into the cell.
Conclusion: The proteins produced in prokaryotic systems were non-glycosylated thus they had different physicochemical properties in comparison with the natural form. So, the production of IFNβ protein in human cell line under strong promoter of selected vector is one of the advantages of this research. Protein studies in this field are targeted for the future studies. |
| first_indexed | 2024-03-12T19:22:44Z |
| format | Article |
| id | doaj.art-5d3acd862db44717840eb251757bd15a |
| institution | Directory Open Access Journal |
| issn | 1027-7595 1735-854X |
| language | fas |
| last_indexed | 2024-03-12T19:22:44Z |
| publishDate | 2015-12-01 |
| publisher | Isfahan University of Medical Sciences |
| record_format | Article |
| series | مجله دانشکده پزشکی اصفهان |
| spelling | doaj.art-5d3acd862db44717840eb251757bd15a2023-08-02T05:03:47ZfasIsfahan University of Medical Sciencesمجله دانشکده پزشکی اصفهان1027-75951735-854X2015-12-0133354169117002029Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This ConstructRaheleh Norouzi0Zohreh Hojati1Department of Biology, School of Science, University of Isfahan, Isfahan, IranAssociate Professor, Department of Biology, School of Science, University of Isfahan, Isfahan, IranBackground: Interferon beta (IFNβ) is one of the important cytokines expressed in response to stimulating factors such as antigens and plays roles in immunity and inflammatory process. In present study, the expression level of IFNβ-1a was examined in HEK293 cell line using real-time polymerase chain reaction (Real-Time PCR). Methods: IFNβ gene sequence was amplified using specific primers contained KpnI and BglII restriction site from pSVMdhfr-IFNβ plasmid as template. It was cloned in similarly digested pBud.CE4.1 linear vector. Construction of recombinant plasmid was verified via restriction fragment length polymorphism (RFLP) analysis, colony PCR and gene sequencing. Recombinant plasmid was transformed into competent Escherichia coli Top10 cells finally. After amplification, recombinant plasmid was purified and transfected into HEK293. At last, RNA extraction, cDNA synthesis and analysis of expression level of gene were performed using Real-Time PCR method. Findings: IFNβ gene was expressed under eEf1a promoter in HEK293 successfully. The expression level of target gene was increased 79.9 times in comparison with the control via transfection. Transfection of null vector showed 2.87 times elevation of target gene expression in response to the alien genome entered into the cell. Conclusion: The proteins produced in prokaryotic systems were non-glycosylated thus they had different physicochemical properties in comparison with the natural form. So, the production of IFNβ protein in human cell line under strong promoter of selected vector is one of the advantages of this research. Protein studies in this field are targeted for the future studies.http://jims.mui.ac.ir/index.php/jims/article/view/5030HEK293 cell lineInterferon betapBud.CE4.1 vectorReal-time polymerase chain reaction |
| spellingShingle | Raheleh Norouzi Zohreh Hojati Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct مجله دانشکده پزشکی اصفهان HEK293 cell line Interferon beta pBud.CE4.1 vector Real-time polymerase chain reaction |
| title | Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct |
| title_full | Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct |
| title_fullStr | Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct |
| title_full_unstemmed | Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct |
| title_short | Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct |
| title_sort | construction of pbud ce4 1 ifnβ human beta interferon and analysis of its expression level in transfected hek293 cell via this construct |
| topic | HEK293 cell line Interferon beta pBud.CE4.1 vector Real-time polymerase chain reaction |
| url | http://jims.mui.ac.ir/index.php/jims/article/view/5030 |
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